Method of treating hemolytic streptococci and the resultant preparation containing the same

ABSTRACT

BY ADDING CEPHALOSPORIN C OR CYCLOSERINE TO A LIVING CELL SUSPENSION OF HEMOLYTIC STREPTOCCI HAVING ANTITUMOR ACTIVITY IN A SUSPENSION MEDIUM TO TREAT THE CELLS WITH THE ANTIBIOTIC, IT IS POSSIBLE TO ELIMINATE TOXICITY WITH INCREASING ANTI-TUMOR ACTIVITY TO MAKE THEM USEFUL ANTITUMOR PREPARATIONS.

United States Patent METHOD OF TREATING HEMOLYTIC STREPTO- COCCI AND THE RESULTANT PREPARATION CONTAINING THE SAME Shigeo Suzuki, Takao Note, and Yoshio Takagaki, Tokyo, Sakae Wada, Ageo, and Akihiro Yamamoto, Haruki Ogawa, Takashi Matsuno, Hiroshi Okazaki, and Yutalra Sugawara, Tokyo, Japan, assignors t0 Chugai Seiyaku Kabushiki Kaisha, Tokyo, Japan No Drawing. Continuation-impart of application Ser. No. 694,854, Jan. 2, 1968. This application Mar. 15, 1971, Ser. No. 124,445

Claims priority, application Japan, Jan. 20, 1967, 42/3,651; Sept. 18, 1967, 42/59,387 Int. Cl. C12k 3/00 US. Cl. 424-93 9 Claims ABSTRACT OF THE DISCLOSURE By adding cephalosporin C or cycloserine to a living cell suspension of hemolytic streptococci having antitumor activity in a suspension medium to treat the cells with the antibiotic, it is possible to eliminate toxicity with increasing anti-tumor activity to make them useful antitumor preparations.

CROSS-REFERENCE TO RELATED APPLICATION This is a continuation-inpart application of parent copending application Ser. No. 694,854, filed on Jan. 2, 1968, now abandoned in favor of the present application.

This invention relates to a method of increasing the anti-tumor activity and reducing toxicity, and also reducing virulence (i.e., pathogenicity) where it exists, of Streptococcus hemolyticus; and to preparations obtained thereby.

It is already known that certain living cells of Streptococcus hemolytiCus have anti-tumor activity. However, it is extremely dangerous to use living cells in the amount required for treating a tumor, since this bacterium is quite toxic and is also a pathogen of erysipelas, etc. It has been reported recently that anti-tumor activity increases When cells of Streptococcus hemolyticus having anti-tumor activity are incubated at about 37 C. for a short time in a medium containing penicillin of a comparative high concentration (Gann vol. 55, pp. 233 236, 1964). -It has been also known that toxicity is decreased and anti-tumor activity further increased when the suspension incubated about at 37 C., above-mentioned, is further subjected to heat treatment at about 45 C. for a short time (The Japanese Journal of Experimental Medicine, vol. 36, pp. 161-174, 1966).

While such a treatment using penicillin can increase anti-tumor activity of Streptococcus hemolyticus and decrease toxicity thereof, the preparation obtained by this method cannot be administered to patients who are susceptible to anaphylactic shock by penicillin. This is an important problem because of recent increases in the numbers of persons sensitive to penicillin as a result of its wide scale use in treatment of various kinds of diseases. Furthermore, because penicillin and protein are easily combined under alkaline conditions, penicillin protein complex causes a rise in the formation of antibodies leading to the development of allergies.

Investigations have therefore been carried out to find means of using Streptococcus hemolyticus in the treatment of tumors whereby such allergies, posing the danger of anaphylactic shock, donot occur and anti-tumor activity effectively increases. It has now been found that Patented Apr. 24, 1973 'ice cephalosporin C and cycloserine can be used in place of penicillin because they increase the anti-tumor activity of the cells of Streptococcus hemolyticus better than penicillin, while the increase cannot be observed in streptomycin and many other antibiotics and chemotherapeutic agents. Further, toxicity and virulence, where it exists, are also reduced when the cell suspensions of Streptococcus hemolyticus with cephalosporin C or cycloserine incubated at about 37 C. is further subjected to heat treatment at about 45 C. for a short time.

According to the present invention, there is provided a new anti-tumor preparation and a new method for treating the cells of Streptococcus hemolyticus which comprises adding cephalosporin C or cycloserine to a cell suspension of Streptococcus hemolyticus in a suspension medium to make the concentration greater than 3 10 moles/ml. and incubating the mixture at 30-38" C. and then preferably treating it at 38-50 C.

Illustrative strains of Streptococcus hemolyticus which may be used in this invention are:

Streptococcus hemolyticus Su (ATCC 21060) Streptococcus hemolyticus 'Sv (ATCC 21059) Streptococcus hemiolytlcus C-203 S (ATCC 21546) Streptococcus hemolyticus Blackmore (ATCC 21548) Streptococcus hemolyticus (ATCC 12202) These Streptococcus hemolyticus may be cultivated, for example, in a meat-infusion broth or a medium mainly consisting of yeast extract, and the cells recovered from the culture broth are suspended in a suspension medium, e.g. Bernheimers Basal Medium (composition: 675 mg. of maltose, 6 ml. of 2% aqueous solution of potassium dihydrogen phosphate adjusted to pH 6.9- 7.0 with sodium hydroxide, 12 ml. of 2% aqueous solution of magnesium sulfate heptahydrate, 66 ml. of distilled water; hereinafter referred to as BBM), distilled water, physiological saline solution, phosphate-buffered solution, phosphate buffered saline solution or any other well known suspension medium.

Cephalosporin C and cycloserine may be directly added to the cell suspension or alternatively, they may be previously dissolved in the suspension medium prior to suspension of the bacterial cells. The concentration to be added in the cell suspension is preferably greater than 3X10 moles/l. Cephalosporin C used for the present invention may be natural or synthetic, including, for example, cephalothin, cephaloridine, etc.

The cell suspension of Streptococcus hemolyticus containing cephalosporin C or cycloserine is incubated at 30-38 0., preferably about 37 C., for about 10-30 minutes, generally 20 minutes, to increase anti-tumor activity of the cells, and the incubated suspension is preferably further subjected to heat treatment at a temperature of more than 38 C. but less than 50 C. to decrease toxicity of the cells.

The thus obtained cell suspension of Streptococcus hemolyticus is suitable as an anti-tumor preparation, as it is, or it is variously diluted with physiological saline solution etc. on actual application in the treatment of tumors.

The anti-tumor preparation of the present invention is better in anti-tumor activity than that obtained using penicillin and it is further better because of no fear of anaphylactic shock; it is suitable for long-term continuous administration to patients who are hypersensitive to penicillin. Further, the anti-tumor preparation has very low toxicity, and the streptolysin S forming ability of the cells is completely lost particularly when cephalosporin C is used.

Examples and experimental examples of the present invention are offered illustratively.

3 EXAMPLE 1 Nine grams of yeast extract (produced by Ebios Yakuhin Kogyo K.K.) were dissolved in 200 ml. of distilled water, adjusted to pH 7.0 72 with sodium hydroxide and heated at 100 C. for 60 minutes. After cooling, the precipitate formed was removed by filtration and the pH of the filtrate was readjusted with 10% sodium hydroxide. The solution was further heated at 100 C. for 30 minutes and filtered. Distilled water was added to the filtrate to bring it to 300 ml., and the resultant mixture was poured into sterilized flasks and subjected to steam sterilization at a pressure of 1 leg/cm. for 10 minutes.

A culture broth of Streptococcus hemolyticus Su-strain, ATCC No. 21060 (nearly a virulent strain), obtained by cultivating the strain for 20 hours in ml. of meatinfusion broth, was inoculated to 300 ml. of the abovementioned yeast extract culture medium, and statically cultivated at 37 C. for hours. The culture broth was subsequently chilled with ice and centrifuged to obtain cells. Such cells were then washed twice with physiological saline and suspended in 15 ml. of BBM. Absorbance of this cell suspension at 660 m was 7.80.

To 5 ml. of the thus obtained suspension, 1 ml. of 0.258 M cephaloridinc-physiological saline solution was added and the suspension was incubated at 37 C. for 20 minutes. The streptococcal suspension thus obtained was subjected to anti-tumor test. The results are shown in Experimental Example 1 and Table 1 below.

EXAMPLE 2 Example 1 was repeated and the anti-tumor suspension obtained was further heated at 45 C. for 30 minutes. The suspension thus treated showed increased anti-tumor activity over that obtained in Example 1 comparing Experimental Examples 1 and 2. Toxicity was decreased.

EXAMPLE 3 Thirty grams of yeast extract (produced by Ebios Yakuhin Kyogo K.K.) were dissolved in 50 ml. of distilled water, adjusted to pH 7.0-7.2 with 10% sodium hydroxide, and heated at 100 C. for 60 minutes. After cooling, the precipitate formed was removed by filtration, the pH of the filtrate was readjusted with 10% sodium hydroxide solution, and distilled water was added to bring the total volume to 1000 ml. The solution was poured into sterilized flasks and subjected to steam sterilization at a pressure of 1 'kg./cm. for 10 minutes. A culture broth of Streptococcus hemolyticus Su-strain ATCC No. 21060 previously cultivated for 20 hours in 50 ml. of a meatinfusion broth was inoculated in 1000 ml. of the abovementioned yeast extract medium, and statically cultivated at 37 C. for 14 hours. The culture broth was subsequently chilled with ice, centrifuged to obtain cells and the thus obtained cells were washed twice with cold physiological saline, and then suspended in 50 ml. of BBM. Absorbance of this cell suspension at 660 m was 9.7.

To 5 ml. of the suspension, 1 ml. of 0.258 M cycloserine physiological saline solution was added, and the resultant mixture was incubated at 37 C. for 20 minutes to increase the anti-tumor activity of the cells. Toxicity was decreased. See Tables 3 and 4 for experimental results.

EXAMPLE 4 Example 3 was repeated and the anti-tumor suspension obtained was further heated at 45 C. for 30 minutes. Toxicity was decreased. The anti-tumor activity of the cell suspension was increased over that obtained in Example 3.

EXAMPLE 5 Streptococcus hemolytz'cus Sv-strain ATCC No. 21059 (highly virulent) was cultivated and suspended into BBM following the same procedure as in Example 1. The absorbance of the bacterial cell suspension at 660 m was 7.90.

To 5 ml. of the thus obtained suspension, 1 ml. of 0.258 M cephaloridine-physiological saline was added and the suspension was incubated at 37 C. for 20 minutes as in Example 1, and further heated at 45 C. for 30 minutes as in Example 2. Virulence and toxicity were decreased, and anti-tumor activity increased. See Table 5 for experimental results.

EXAMPLE 6 Streptococcus hemolytt'cus C-203 S strain ATCC No. 21546 was cultivated and suspended in BBM following the same procedure as in Example 1. The absorbance of the bacterial cell suspension at 660 me was 5.00.

To 5 ml. of the thus obtained suspension, 1 ml. of 0.258 M cephaloridine-physiologieal saline solution was added and the suspension was incubated at 37 C. for 20 minutes as in Example 1, and further heated at 45 C. for 30 minutes as in Example 2. Toxicity was decreased, and. anti-tumor activity increased. See Table 6 for experimental results.

EXAMPLE 7 Slreptococcus hemolyticus Blackmore strain ATCC No. 21548 was cultivated and suspended in BBM following the same procedure as in Example 1. The absorbance of the bacterial cell suspension at 660 mu was 8.60.

To 5 ml. of the thus obtained suspension, 1 ml. of 0.258 M cephaloridine-physiological saline solution was added and the suspension was incubated at 37 C. for 20 minutes as in Example 1, and further heated at 45 C. for 30 minutes as in Example 2. Toxicity was decreased and anti-tumor activity increased. See Table 7 for experimental results.

EXAMPLE 8 Streptococcus hemolyticus Sv-strain ATCC No. 21059 was cultivated and suspended in BBM following the same procedure as in Example 1. The absorbance of the bac' terial cell suspension at 660 m was 7.9.

To 5 ml. of the thus obtained suspension, 1 ml. of 0.258 M cycloserine-physiological saline solution was added and the suspension was incubated at 37 C. for 20 minutes as in Example 3, and further heated at 45 C. for 30 minutes as in Example 4. Toxicity and virulence were decreased, and anti-tumor activity was increased. See Table 5 for experimental results.

EXAMPLE 10 Streptococcus hemolytz'cus C-203 S strain ATCC No. 21546 was cultivated and suspended in BBM following the same procedure as in Example 1. The absorbance of the bacterial cell suspension at 660 m was 5.00.

To 5 ml. of the thus obtained suspension, 1 ml. of 0.258 M cycloserine-physiological saline solution was added and the suspension was incubated at 37 C. for 20 minutes as in Example 3, and further heated at 45 C. for 30 minutes as in Example 4. Toxicity was decreased and anti-tumor activity increased. See Table 6 for experimental results.

EXAMPLE 11 Streptococcus hemolyticus Blackmore strain ATCC No. 21548 was cultivated and suspended in BBM following the same procedures as in Example 1. The absorbance of the bacterial cell suspension at 660 m was 8.60.

To 5 m1. of the thus obtained suspension, 1 ml. of Comparative tests were simultaneously run with the 0.258 M cycloserine-physiological saline solution was stfefptococcal P slmllflfly treated P added and the suspension was incubated at 37 C for 20 mum G potassium (HI) dlhydrostreptomycm Sulfate (IV) streptococcal suspension untreated with antibiotics minutes as in Example 3, and further heated at 45 C.

5 and (V) a BBM free from the streptococcal cells but confor 30 minutes as m Example 4. Toxicity was decreased taining the carcinoma cells.

TABLE 1 Streptococcal suspension Survival of mice after- Dilution Cone. of each drug in num- 10 20 30 50 Group Drug added physiological saline an. solution bers days days days days I hi .1- 1 8 27? 212 it? 2}? c 1 on me 08 mg. otenc ml. 0.258

p M) x30 5 4/5 4 5 3 5 X40 5/ 5 4/ 5 2/5 2/5 X 5/5 5/5 5/5 5/ 5 II Penicillin G po ium 1.6Xl0 units/ml. (0.25SM) X 5 5 5 5 X 5/5 4/5 1/ 5 1/5 X 5/5 3/ 5 l/ 5 0/ 5 III Dihydrostreptomycin sulfate 188 mg. (potency)/ml. (0.258 M) g X30 5/5 2/ 5 0/ 5 0/ 5 IV None; Physiological saline only- "$68 g X30 5/5 2/5 0/5 5 V Control (not containing streptococcal cells; BBM only) Nora-Concentration of each drug in physiological saline water was adiusted so that it became an eqni-molar concentration with Penicillin G otassium 1.6)(10 units/ml. The calculation was performed by presuming Penicillin G potassium 1 mg.=l667 units. and 1.6Xl0 units/ml.=0.258 M.

orepvfir, the concentration of dihydrostreptomycin was calculated on the basis that 3 moles of sulphite were present for 2 moles of the dihydrostreptomycin ase.

and anti-tumor activity increased. See Table 7 for ex- EXPERIMENTAL EXAMPLE 2 perimental results. (ANTI-TUMOR TEST) EXAMPLE 12 Stre tococcus hemol ticus ATCC No. 12202 was culti- 30 Streptococcal suspension obtained in Example 2 vated Suspended g the Same p Was (hinted-2'0 fold, fold, and cedure as in Example 1. The absorbance of the bacterial BBM .contammg 0'043 m.01e/ of cephalondme' cell Suspension at 660 my was Anti-tumor test was earned out following the procedure To 5 of the thus obtained Suspension 1 mL of r as used in Experimental Example 1, except using 8 mice 0.258 M cycloserine-physiological saline solution was for each groupadded and the suspension was incubated at f Comparative tests were simultaneously run with the 20 minutes as in Example 3, and f th heated at c streptococcal suspenslon similarly treated with (II) penifor 30 minutes as in Example 4. Toxicity was decreased 3111111 G Potasslum and a BBM free from the p and anti-tumor activity increased. See Table 8 for ex- 40 coccal cells but containing the carcinoma lls. perimental results. The results are shown in Table 2.

TABLE 2 Streptococcal suspension Survival of mice after- Dilution Cone. of each drug in physiological nnm- 10 20 30 Group Drug added saline solution bers days days days days iit 242 242 2;: as I Cephalm'idme 108 mg. (potency)lm1. (0.258M)... X 8/8 8/8 7/8 7/8 X80 8/8 7/8 5/8 5/8 X20 8/8 8/8 8/8 8/8 11 Penicillin Gpotassinm 1 7 115x10 units/ml. 0.253 M) g2 X80 8/8 3/8 l/8 1/8 1H Control (not containing streptococcal cells; BBM only) 8/8 0/8 0/8 0/ 8 EXPERIMENTAL EXAMPLE 1 EXPERIMENTAL EXAMPLE 3 (ANTI-TUMOR TEST) The streptococcal suspension obtained in Example 1 i was diluted 10 told, 20 fold, 30 fold and 40 fold with st'eptowccl hemolytlcus 5mm ATCC 21060 was BBM containing 0 043 mole/l of cephaloridine 60 cultivated by the procedure shown in Example 3 and bacf ascites W ainected the 8th day after i i niii ii pfl iy sii i giiz l s aiir iy solution of cycloserine, fate:eatiftt zhatsdztassistant 11122;: e was a e to m o t is ce suspension, and t on in- :as at stains: stasis; tests; at g 81011 was furt er eate at or minutes as in gl s lzzi e g iz Egg 22 agi gfii gg ai i g 22: 323 Example 4, and diluted with BBM to 2 fold, 4 fold and 8 fold and mediatl u d th a t'- um eri- 6X10" per ml. Each l ml. of this carcinoma cell snspen- 1m 6 y Se m e n It or exp (A) Sample preparation sion was mixed with each 3 ml. of the above-mentioned ment' (B) Anti tumor experiment suspensions incubated at 37 C. for 90 minutes, and 0.5

ml. of each the mixed solution was subsequently intra- To ddY strain male mice (average body weight about peritoneally injected into mice (ddY strain, average body 20 g.), to which Ehrlich ascites carcinoma cells (Mouse weight: about 20 g.), each group consisting of 5 mice. ascites on 8th day after inoculation) had been intra- Survival number of each group was observed. The results peritoneally inoculated in a quantity of 10 cells per are shown in Table 1. mouse, the above-mentioned BBM diluted suspension were intraperitoneally injected 0.1 ml. per mouse, initiated The results of the tests are shown in Table 5.

TABLE Streptococcal suspension Survival of mice after- Dilution Cone. of each drug in num- 20 30 50 Group Drug added physiological saline solution bers days days days days I Cycloserine 26.4mg. (potencyflml. (0.258M) X4 8/8 7/8 6/8 5/8 8 8/8 6/8 5/8 Cephaloridine 108mg. (potencyflml. (0258M) 4 8/8 8/8 8/8 7/8 X8 8/8 7/8 5/8 5/ III Penicillin G potassium 1.6)(10 units/ml. (0.258M) {X4 8/8 7/8 6/8 5/8 X8 8/8 3/8 2/8 2/8 IV Control (not containing streptococcal cellsBBM only) 8/8 0/8 0/8 0/8 24 hrs. after the inoculation, once a day for a period of EXPERIMENTAL EXAMPLE 6 5 days. Eight mice per group were used and survival (ANTI-TUMOR TEST) number of each group was observed. The results are snepmocus hemogyticus 203 5 Strain A NO shown in Table 3. 21546 was cultivated by the procedure shown in Example TABLE 3 Streptococcal suspension Survival of mice after- Dilution Cone. of each drug in num- 10 50 Group Drug added physiological saline solution bets days days days days I Cycloserine id-4mg. (poteney)/ml. (0.258 M). 3g 242 S52 11 Penicillin G potassium 1.6X10 units/ml. 0.258 M) 3g 5g 3;; III Dihydrostreptomycin sulfate 188 mg. (potency)/ml. (0.25s M gig 3;; 3;: Control (not containing streptococcal cells; BBM only) 8/8 0/8 0/8 0/8 EXPERIMENTAL EXAMPLE 4 6, and the cells were suspended in BBM and treated with Anti-tumor test was repeated in the same manner as cephaloridine or cycloserine by the procedure of Example in Experimental Example 3, except that the concentra- 6 or 10, respectivelytion of cycloserine and penicillin G potassium are adjusted 26.4 mg./ml. and 4.4 10 units/ml. Which is equivalent concentration therewith, respectively.

Anti-tumor tests were carried out by the method described in Experimental Example 3.

The results are shown in Table 4. Comparative tests were simultaneously run with the TABLE 4 Streptococcal suspension Survival of mice aiter- Dilution Cone. of each drug in num- 10 20 30 Group Drug added physiologicalasaline solution bers days days days days 1 Cycloserine 26.4 mg- (poteney)/ml- (0.25s 3% $43 2 11 Penicillin Gpo I 4.4Xl0 units/ml. (26.4 m ./m1. 24g 34g III Control(notcontainingstreptococcalcells;BBMonly) 8/8 0/8 0/8 0/8 EXPERIMENTAL EXAMPLE 5 50 streptococal suspension similarly treated with penicillin G potassium Streptococcus hemolyticus Sv-strain ATCC No. 21059 The results of the tests are shown in Table 6.

TABLE 6 Streptococcal suspension Survival of mice after- Dilution Cone. of each drug in num- 10 20 30 50 Group Drug added physiological saline solution bers days days days days I Cycloserine 26.4 mg. (potencyflml. (0.258 M) 4 8/8 6/8 4/8 4/8 8 8/8 4/8 3/8 3/8 II Cephalon'dine... 108 mg. (potency)/ml. (0.258 M) 3/3 2;: 3/2 243 III Penicillin G potassium 1-6X105 units/m1. (0.258 M) IV Control (notcontainingstreptoeoccalcells;BBMonly)-. 8/8 0/3 0/3 0/8 was cultivated by the procedure shown in Example 5, EXPERIMENTAL EXAMPLE 7 and the cells were suspended in BBM and treated with (ANTI-TUMOR TEST) cephalofidim 0T fiyclosel'ine y the Procedure of Exam Streptococcus hemolyticus Blackmore strain ATCC No. P 5 0f 9, respectively 21548 was cultivated by the procedure shown in Example Anti-tumor tests were Carried ut by t meihOd 7, and the cells were suspended in BBM and treated with scribed in Experimental Example 3. cephaloridine or cycloserine by the procedure of Ex- Cornparative tests were simultaneously run with the ample 7 or 11, ti l streptococcal suspension similarly treatfid With penicillin Anti-tumor tests were carried out by the method de- G potassium, scribed in Experimental Example 3.

Comparative tests were simultaneously run with the streptococcal suspension similarly treated with penicillin G potassium.

The results of the tests are shown in Table 7.

injected to each ddY mouse (average body weight about 20 g.) and the number of mice dying within 7 days was observed. The results obtained by using 10 mice for each group are shown in Table 10.

TABLE 7 Streptococcal suspension Survival of mice after- Dilution Cone. of each drug in num- 10 20 30 Group Drug added physiological saline solution bers days days days days I cycloserine. 26.4 mg. (potency)/ml. (0.258 M) {X4 8/8 6/8 6/8 5/8 8/8 5/8 4/8 3/8 II ce haloridine 108 mg. (potency)/m1. (0258M) 4 8/8 7/8 6/8 6/8 X8 8/8 6/8 4/8 4/8 111 Pencillm G potassium 1.6X105/m1. units (0.258 M) 4 8/8 6/8 5/8 5/8 X3 8/8 5/8 3/8 3/8 IV Control (not containing streptococcal cells; BBM only) 8/8 /8 0/8 0/8 EXPERIMENTAL EXAMPLE 8 Table 10 (ANTI'TUMOR TEST) Treating drug: Number of dead mice Streptococcus hemolyticus ATCC No. 12202 was cultig i ii 0/10 vated by the procedure shown in Example 8, and the cells i m potassmm 0/10 we suspended in BBM and treated with cephaloridine It Out usmg drug 8/10 EXPERIMENTAL EXAMPLE 11 (STREPTOLYSIN- 25 S FORMING ABILITY TEST) One ml. of sodium ribonucleate solution (concentrati 8%) in BBM was added to 1 ml. of a treated cell suspension obtained in the same manner as that Experimental 30 Example 9, incubated at 37'C. for 2 hours, and after centrifugation, hemolytic activity of the supernatant was TABLE 8 Streptococcal suspension Survival of mice after Dilution Cone of each drug in num- 10 20 30 Group Drug added physiological saline solution bers days days days days I cycloserine- 26.4 mg. (potency)/ml. (0.258 M). X4 8/8 5/8 5/8 4/8 8 23/8 4/8 3/8 3/8 [T Cephalnndme 108 mg. (potency)lml. (0.258 M) {X8 8/8 6/8 5/8 5/8 X8 8/8 4/8 4/8 3/8 III Pemclllm G potassium 1.6)(10 U-DltS/m (0.258 M) {X4 8/8 5/8 4/8 4/8 X8 8 8 3 8 3 8 2 8 IV Control (not containing streptococcal cells; BBM only). /3 o/g 05 0/3 EXPERIMENTAL EXAMPLE 9 (TOXICITY TEST) Physiological saline solutions of cephaloridine or penicillin G potassium were added in a ratio of 0.2 ml. each to 1 ml. of a BBM suspension of cells of Streptococcus hemolyticus Su-strain ATCC No. 210 60 obtained in the same manner as in Example 1 and incubated at 37 C. for minutes, and then heated at 45 C. for minutes.

0.1 ml. of this treated suspension Was simultaneously injected on the back of each ddY mouse (average body weight about 20 g.) and the number of mice dying within 24 hours was observed.

The results obtained by using 10 mice for each group are shown in Table 9.

Table 9 Treating drug: Number of dead mice cephaloridine 0/ 10 Penicillin G potassium 0/ 10 Without using drug 8/ 10 EXPERIMENTAL EXAMPLE 10 (TOXICITY. TEST) 0.2 ml. of a physiological saline solution of cycloserine or penicillin G potassium having the same concentrations as those used in Experimental Example 3 were added to 1 ml. samples of BBM cell suspension of Streptococcus hemolyticus Su strain ATCC No. 21060 obtained in the same manner as in Example 3 and heated at 37 C. for 20 minutes and subsequently heated at C. for further 30 minutes. 0.1 ml. of this suspension was intraperitoneally determined by using rabbit erythrocytes according to t convention method.

The results of the tests are shown in Table 11.

Table 11 Streptolysin-S formi ability-hemoly 8 sis It will be obvious to those skilled in the art that various changes may be made without departing from the sco of the invention and the invention is not to be consider limited to what is described in the specification.

What is claimed is:

pe ed 1. A method for treating the cells of Streptococcus hemolyticus to increase anti-tumor activity and decrease toxicity, which comprises: adding an antibiotic selected serine to a cell suspension of Streptococcus hemolyticus from the group consisting of cephalosporin C and cycloa suspension medium to make the concentration of said antibiotic at least 3 10 moles/L, incubating the mixture at 3038 C. and further heating the mixture at a 0 temperatuure of more than 38 C. but less than 50 C.

2. A method claimed in claim 1, in which Streptococcus hemolyticus is selected from the group consisting of Streptococcus hemolyticus Su-strain ATCC No. 21060; Streptococcus hemolyticus Sv-strain ATCC No. 21546; Streptococcus hemolyticus C-203 S ATCC No. 21546; Strep- 1 1 tococcus hemolyticus Blackmore ATCC No. 21548; and

Streptococcus hemolyticus ATCC No. 12202.

3. A method claimed in claim 1, in which the first incubation period is 10-30 minutes and the further heating period is 20-60 minutes.

4. A method claimed in claim 1, in which the first incubation period is 20 minutes and the further heating period is 30 minutes. I

5. A method claimed in claim 1, in which the suspension medium is Bernheimers Basal Medium composed of 675 mg. of maltose, 6 m1. of 2% aqueous solution of potassium dihydrogen phosphate adjusted to pH 6.9-7.0 with sodium hydroxide, 12 ml. of 2% aqueous solution of magnesium sulfate heptahydrate and 66 ml. of distilled water.

6. A method claimed in claim 1, in which the suspension medium is phosphate buifered solution.

7. A method claimed in claim 1, in which the suspension medium is phosphate buffered saline solution.

12 8. A method claimed in claim 1, in which cephalosporin C is selected from the group consisting of natural cephalosporin C, cephalothin and cephaloridine.

9. The product produced according to the method of claim 1.

ALVIN E. TANENHOLTZ, Primary Examiner M. D. HENSLEY, Assistant Examiner US. Cl. X.R. 195-96, 114 

